PROJECT SUMMARY/ABSTRACT Development of an effective cryopreservation technology for human tissues is necessary to address critical biomedical needs by solving a worldwide shortage of transplantable human tissues and shelf-life problems of engineered tissue constructs that hinder mass production, storage, distribution, and safety/quality control. Moreover, the development of a reliable tissue preservation method would allow better tissue matching toward increased overall success rates and reduced burden of immunosuppression. By enabling viable banking of diseased tissues, an effective preservation method would also advance biomarker discovery and drug testing, and thus personalized medicine toward new effective therapies of devastating diseases such as cancer. Current preservation strategies are inadequate for multicellular complex tissues. The overall goal of the proposed research is to meet the critical need for effective, widely applicable tissue preservation technology by developing a novel approach to vitrification, a cryopreservation strategy involving the solidification of liquid in a glass-like state using high concentrations of cryoprotective agents (CPAs). Vitrification is a promising technique, but CPAs have inherent cytotoxicity. In fact, chemical toxicity of CPAs is considered to be the main barrier to successful cryopreservation of complex tissues. Reduced CPA concentrations with increased cooling and warming rates ameliorate toxicity, but sufficiently fast cooling and warming rates are difficult to achieve in tissues and may lead to extreme thermal stresses, resulting in tissue cracking. To overcome the limitations of current tissue preservation approaches, we propose to address chemical toxicity of CPAs using an interdisciplinary approach of targeted interventions and biophysical modeling. Our preliminary studies revealed that the primary toxicity of 1,2-propanediol (PROH), a preferred CPA, occurs through mitochondrial Ca2+ overload. We were able to completely prevent PROH?s toxicity in mouse oocyte, human fibroid and mouse brain tissue models by using inhibitors of mitochondrial Ca2+ uniporter. Recently, we have also mathematically optimized a procedure to add high CPA concentrations to endothelial cells with minimal cytotoxicity. Based on these encouraging preliminary data, our central hypothesis is that an interdisciplinary approach combining targeted inhibition of CPA toxicity with mathematical modeling and optimization can enable employment of high CPA concentrations, leading to a versatile tissue vitrification method applicable to diverse tissues. The proposed vitrification approach is innovative, because it circumvents the main barrier to the use of high CPA concentrations (CPA toxicity), enabling better suppression of ice nucleation and devitrification; other innovations include the development of a novel vitrification medium that can block ice crystal growth through synthetic polymers and mitigate free radical damage by optimized composition of antioxidants and the use of a novel biophysics-based mathematical optimization strategy to identify minimally toxic protocols for vitrification. The proposed research is expected to pave the way for viable banking of human tissues.